Correlating structure, function and dynamics of RNA requires fundamental understanding of structural basis in RNA folding. The objectives of this proposal are to elucidate the structural basis of RNA folding and how ribozymes fold during transcription, a mimic of folding behavior in the cell. The RNA component of bacterial ribonuclease P will be used. Four crystal structures of this ribozyme have been determined since 2003, enabling us to make fundamental advances in understanding the folding of this large ribozyme. Aim 1 will reveal the major events along the folding pathway of a large RNA by the structural determination of folding intermediates at the residue-level resolution using an integrated approach of solution studies/molecular modeling/cryo-Electron Microscopy. This first of its kind look at RNA folding pathways will offer unprecedented insights on the order of structure formation, the interplay between core and peripheral structures, and the role of metal ion-RNA interactions that guide folding and modulate stability. Aim 2 will compare structures of key folding intermediates among homologous RNAs that have the same core, but distinct peripheral structures. Aim 3 will identify mechanisms for altering folding during transcription, in particular, how prominent pause sites introduced by bacterial RNA polymerase affects folding and the assembly of a RNA-protein complex. This broad research program will reveal the structural basis of RNA folding, and the evolutionary and mechanistic link between RNA folding and RNA polymerase.Narrative Ribonucleic acids (RNA) are not only messengers for protein synthesis, they also play essential roles in regulating gene expression upon folding into elaborate structures. This project aims to uncover the structural and mechanistic basis of RNA folding in order to reveal relationships of RNA folding and function.